Process for production of citric acid by fermentation



Patented Dec. 12, 1944 UNITED) STATES- PATENT arms rnocsss PRODUCTION-GEM Acmh FEBMIINTATION William ,Eise'n man and Max D. Bliunenleld, New

York, N. 1., asslgnorl, by means assignments,

to Ilcyden Chemical Corporation, New York, N. 1., a corporation of Delaware No Drawing.

Application November 7, 1941, Serial No. 418,184

,scmma (Cl. 195-36) as blackstrap molasses, beet molasses, and Hytest cane molasses, since these originally contain amounts of nitrogen and potassium exceeding the limits for these media. This is evident when the compositions of .Curries and Doelger and Prescotts media are. compared with the total, ash, nitrogen and potassium present in twenty per cent solutions of various carbohydrate raw materials, as set forth below.

TABLE II Nitrogen and potassium content of diluted-raw materials fermentablc carbohydrates) Raw material Sucrose andlnvert sugar Ash Nitrogen Potassium Per cent Per cent Per cent Refined sugar.-. Trace Trace. Raw sugar- 0.04 to 0.08.. Trace to 0.012. 0.012 to 0.016;

C }o.5z to o 51.- 0.029 to 0.057.- 0.115 to 0.230.

e mum, mom! }s.a to 4.2.-.. 0.167 to 0.250-- 0.75 to 0.92. Beet molasses 2.5 to 5.1..-- 0.655 to 0.900" 1.15 to 1.3L

It has longbeen recognized that in fermenta- It should be noted, for example, that blacktion processes mold fungi require for their metabolism certain nutrient substances which are chemically independent of, and unrelated to, the carbohydrate undergoing fermentation. Such nutrientsmay be salts or other compounds which supply nitrogen, potassium, phosphorus, sulfur, zinc and magnesium in usableform to the fungi.

It is important in, preparing an emcient, controlled fermentation medium to add all the necessary nutrients, and to add them in the correct weight ratio to each other and to the carbohydrate material present. Currie (.l. Biol. Chem: 31, 15-37 (1917)) and Doelger and Prescott (Ind. Eng. Chem, 26, 1142 (1934)) claim to have prepared such media most favorable for the citric fermentation by Asperpillus niger. The cornpositions of their media ingrams per 1000 cc. of solutions are as follows:

These two very similar media can be easily -prepared from relatively pure but expensive raw 'sugar, and refined sugar.

However, it is impossible to prepare them from the more'impure, but

/- commercially practical, starting materials such strap molasses having 20% sucrose contains at least 0.167% nitrogen and 0.75% potassium, whereas the medium of Doelger and Prescott, which has 15% sucrose permits no more than 0.117% nitrogen and0.037% potassium, based on 20% sugar.

The molasses type of raw material may contain varying percentages of impurities in addition to,. or. as sources of, the relatively high amounts of nitrogen and potassium such as sulfates, chlorides, nitrates of ammonium, calcium, sodium and potassium; amino acids, proteins in various stages'of degradation, unfermentable, sugars such as glutose, organic acids such" as acetic and lactic or their salts, and traces. of heavy metals such as iron copper, nickel and chromium.

Since it is uneconomical to purify these raw materials which are by-products of the sugar industry, many investigators have tried to determine the conditions under which mold fungi would ferment them in their impure state to citric acid. We have now discovered that if sufiicient additional amounts of nitrogen and potas slum are provided in nutrient, form and in the. ratio of 1 nitrogen to 1.35-l.5 potassium, mold fungi will grow more rapidly, and will give high yields of citric acid and high production per fermenting unit. I

This isa surprising discovery, as molasses already contains more nitrogen and potassium I than the media of Currie and of Doelger and Prescott. Furthermore, it has always been claimed'in the literature that large amounts of nitrogen .encourageexcessive growth of the Cl, S04: in objectionablequantiti'es is avoided; The use of urea as a cheap source of organic nitrogen in fermentation is well known. Also, the necessity for the presence of potassium-salts in the fermenting mediumis well known. These facts are in themselves separately recognized.

However, we have found in our work on the citric acid fermentation that these ,twoessential' components must be supplied to the medium in nutrient form and in definite "and fixed ratio to each other. When urea and'potassiu'm acetate sugar, were added:

' obtained.

Americanjtype culture collection. Fermentation was carriediout. for :6 days betweenj 2 8 nd C. A56%. yield (2 82 gr);offcitricz -acld wvas T011300 g. .ofmolasses containing ,BS Oqg-of.

' Grams Urea (4.2 g. of:N) 9 Glacial acetic,:acid 12.6 Kz'CO3.2I-IzO-;(6.l.;g,,of.-rK 13.6 KI-IaPQi .115 1M'g'SQ4 2 0115 ZIISQq 0.10

- to: 3900 'cc. (14.*.0 '-B.).' .Inoculation".was.. carried (or another fattyacid salt of potassium). is supplied, and at certain fixed ratios, results are obtained which are far superior to any hitherto known. So far as we know, no one has used these two essential components together as we have done. The resulting fermentation, takes less time and gives yields of citric acid'of greater.

purity and much higher percentage.

With this quantity and ratio of nutrient The resulting-:- solution wasldiluted with water out in the usual manner h spores from; strain collection. 'E'ermentation was caricaturi'or 7 or citric a i ves emes. macaw .1300 g. of molasses. were f treated .initbe same manner as .described in. Eigamplelfl, except: that materials supplying (the additional amounts of effective nitrogen and effective ,potas'siurn' (urea, acetic acid, potassium carbonate) were omitted.

nitrogen and nutrient potassium, there is a very rapid growth of themycellium. The production of acid starts earlier in the life cycle of theorganism. There is no slowing down due to abnormal rise of temperature. This quantity and ratio of nutrient nitrogen and nutrient potassium enables the organism to ignore completely the presence of other nitrogen and potassium com- [Little or no citric.acidiwas termed.

"When one comparesithe yieldobtainediin Example 1 with yields reportedfby; Doelger and Prescott (compare (a) ;a;1 1d;(b).'I,able .I-II) it is app'arentithat, using" a .cr'uder, sugar;-;wei-have increased the average. yieldiof citric acid. by almost ponents that may be present as impurities in the carbohydrate materials.

It should be recognized at this point that in:

addition to nitrogen and potassium? magnesium, zinc, phosphorus and sulfur must be present in the medium in the conventional forms and amounts.

The following examples describe two of the I various ways in which the principle of our invention may be employed. his to be understood, however, that such examples are purely illustrative, and are not to ,be' construed as a limitation on the invention, since many successful variations maybe made as long as the indicated amounts of, and ratio between, nitrogen and potassium are maintained.

EXAMPLE 1 To a solution of 500 g. of sucrose (commercial refined) in 1000 g. of water, were added:

/ ,Grams' Urea (4.2 g. ofN) l 9 Glacial acetic acid 12.6 K2CO3.2H20(6.1 g. of. K) 13.6 KHzPO4 1.5 MgSO4.7HzO 0.75 ZHSO4.7H2O 0.05

. The ratio of nitrogen to potassium, namely 4.2 to 6.1, is about 1 to'l.5. The acetic acid in reacting with the KzCOaZHzO produces 15.32 g. of potassium acetate.

The resulting solution was diluted to 2500 cc.

and was inoculated in the usual mannerwith. Aspergillusniger spores from the strain #1015 Furthermore, from a commercial standpoint, it is justas importanttonpit-that. we havealso been able to increase theoutput of citric acid per' pan :more than times that ofrpoelger and Prescottwithout loweringjthe. yield. (Compare (alan'dfe') of the followingtable):

TAsrrIIII (From page 363, Industrial Microbiology," .by Prescott- 6: Dunn) Doelger. and Prescott Medium (Pan 25 x,-33cm.)

Medium Yield volume Orig- Su- Final Citric citric surface inal crose "mmme acid per acid acid in area ratio, volume per pen in cc cc. r {m percent cc. per in co. in g. in g. 9% p of sugar sq. cm. e

a 2. 45 2,000 280 I 1, 810. 6. 35 114. 9 4]. 0 b 2. 20 1, 800 252 1, 620 7. 06 114. 2 45. 3 c 2. 08 1, 700 238 1, 490 7. 40 110.3 46. 3 c" 1.83 1,500 210 1,310 7. 75 110.12 48.3 d, 1.22 1, 000- 8; 80. 6 40.0

Duration days.

Our-medium as shownln Example 2 (Pen 25 r at cm.)

Medium Yield volume Orig- Su Find Citric citric surface inal crose volume acid per acid acid in area'ratio, volume per pan in cc 10000 an rcent cc. per in 00.. in g. in g oisugar sq. cm. used a 5. 15 4, 375 875 4, 000 12. 43 497 56. 8 f 4. 20 3, 750 750 4, 200 10. 45 439 58:6 0 3.00 2. 500 500 a, 000 9.40 232 56.4

Duration 7 to!) days.

Referring to Table. III, larger volumes may be used under (e);, (l) andig) thanin (a), (b), and

(0), because it is possible with our medium to fermentthe larger volume or solution with the same exposed area, without any decrease in yield. Doelger and Prescott were not able to use larger volumes with their medium without a drop in the yield.

These improvements result from adding to the medium the proper amounts of nutrient nitrogen and potassium in proportion to the amount of fermentable sugars present, and in the proper ratio to each other. We do not advance any theory to explain the superior results obtained in spite of inferior raw materials, but only reiterate that nitrogen and potassium in the form, amounts and relative ratios in which we add them, constitute essentially the necessary nutritive material for rapid initial growth of the fungi,

2. The process for producing citric acid, which comprises fermenting a sucrose medium containand rapid production of citric acid in the presence of impurities which would otherwise be detrimental.

Having thus described our invention, what we claim as new and desire to secure by Letters Patent is:

1. The process for producing citric acid, which comprises fermenting a sucrose medium containing from 9 grams to 15 grams of an organic nitrogen-containing compound and 14 grams to 26 grams of a potassium salt of an organic acid per 2500 cc. of mixture containing 20% sucrose, with a citric acid producing fungus.

ingfrom 9 grams to '15 grams of an organic nitrogen-containingcompound and 14 grams to 26 grams of'a potassium salt of an organic acid per 2500 cc. of'mixture containing 20% sucrose, with Aspergillus niger.

3. The process for producing citric acid, which comprises fermenting a sucrose medium containing from 9 grams to 15 grams of an organic nitrogen-containing compound and 14 grams to 26 grams of potassium acetate per 2500 cc. of miirture containing 20% sucrose, with agcitric acid producing fungus.

4. The process for producing citric acid, which comprises fermenting a sucrose medium contain-,- ing from 9 grams to 15 grams of urea and 14 grams to 26 grams of a potassium salt of an organic acid per 2500 cc. of mixture containing 20% sucrose, with a citric acid producing fungus.

5. The [process for producing citric acid, which comprises fermenting a sucrose medium containing from 9 grams to 15 grams of urea and 14 grams to. 26 grams of potassium acetate per 2500 cc. of mixture containing 20% sucrose, with a citric acid producing fungus.

WILLIAM vEISENMAN. MAX D. BLUMENFELD. 

